The Notch signal transduction pathway controls cell fate decisions important for neurogenesis and hematopoiesis and is involved in various cancers including leukemia. Signaling results in the proteolytic release of the intracellular domain (ICD) of the Notch receptor, a dedicated transcriptional co-activator of CSL enhancer-binding proteins. Our lab recently developed a cell-free system that recapitulates Notch transcription on a simple model enhancer. These studies revealed that the ICD activates transcription only in conjunction with a second Notch co-activator, Mastermind (MAM). We showed that MAM recruits the CBP/p300 histone acetyltransferase as well as other components of the mediator complex including the kinase, CDK8. Finally, we showed that co-expression with MAM also promotes the phosphorylation and degradation of the ICD in vivo. These findings suggest that MAM may recruit a nuclear kinase to couple activation with turnover of the ICD. Here I propose to investigate the role of MAM-associated co-activator proteins in Notch transcription and the co-ordination of activation with proteolytic degradation of the ICD through the following specific aims: I. Define the conserved properties of the Mastermind family of Notch co-activators, II. Assess whether the protein kinase activity of CDK8 contributes to Notch transcription and/or the turnover of the ICD in vivo and in vitro, and III. Examine the role of the c-ski-interacting protein (SKIP) in Notch transcription in vitro. Through these studies we hope to better understand the Notch transactivation mechanism and its role in the modification and destruction of the ICD and taken together, these studies will advance our understanding of the mechanism by which the Notch enhancer complex induces endogenous target genes during transformation.